Chemicals, drugs and solutions
VGB ((±)-y-vinyl-GABA, C6H11NO2, https://pubchem.ncbi.nlm.nih.gov/compound/vigabatrin) was acquired from Sigma-Aldrich (Merck Ltd., Taipei, Taiwan), GAL-021 was from MedChemExpress (Everything Biotech Ltd., New Taipei City, Taiwan), while DCEBIO (5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one) and TRAM-34 (1-((2-chlorophenyl)-(diphenyl)methyl)-1H-pyrazole) were from Tocris (Union Biomed, Taipei, Taiwan). Unless specified otherwise, the culture media, fetal bovine serum, L-glutamine, and trypsin/EDTA were acquired from HyCloneTM (Thermo Fisher Scientific, Taipei, Taiwan); all other chemicals or reagents were of analytical grade.
The composition of the bathing solution (i.e., HEPES-buffered normal Tyrode’s solution) was 136.5 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.53 mM MgCl2, 5.5 mM glucose, and 5.5 mM HEPES titrated with NaOH to pH 7.4. To measure K+ currents, we backfilled the patch pipettes with an internal solution consisting of 130 mM K-aspartate, 20 mM KCl, 1 mM KH2PO4, 1 mM MgCl2, 3 mM Na2ATP, 100 μM Na2GTP, 0.1 mM EGTA, and 5 mM HEPES adjusted with KOH to pH 7.2 [18, 19]. To avoid the contamination of whole-cell Cl- currents, we replaced Cl- ions inside the pipette solution with aspartate.
In the experiments on recording large-conductance Ca2+-activated (BKCa) channels, a high K+-bathing solution was used, and its composition was 145 mM KCl, 0.53 mM MgCl2, and 5 mM HEPES titrated with KOH to 7.2, and the pipette solution contained 145 mM KCl, 2 mM MgCl2, and 5 mM HEPES titrated with KOH to 7.2. All solutions described above were prepared using demineralized water from a Milli-Q water purification system (Merck, Ltd., Taipei, Taiwan). On the day of use, we filtered the pipette solution and culture medium by using an Acrodisc® syringe filter with a Supor® membrane (Bio-Check; New Taipei City, Taiwan) [20, 21].
Cell preparations
The glioblastoma multiforme cell line (13-06-MG) used was kindly provided by Professor Dr. Carol A. Kruse (Department of Neurosurgery, Ronald Reagan UCLA Medical Center, LA, U.S.A). We routinely grew the 13-06-MG cells at a density of 106/ml in high glucose (4 g/l) Dulbecco’s modified Eagle media (Invitrogen, Carlsbad, CA, USA) which were supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin and 10 μg/ml streptomycin. Cells were maintained at 37˚C in a 5% CO2 incubator as monolayer cultures and then sub-cultured weekly, and fresh media was added every 2-3 days to maintain a healthy cell population. We also verified glial cells by identifying glial fibrillary acidic protein, which is a cytoskeletal protein.
To estimate concentration-dependent inhibition of VGB on the open-state probability of IKCa channels, 13-06-MG cells were bathed in normal Tyrode’s solution containing 1.8 mM CaCl2, and each cell examined was voltage-clamped at -80 mV relative to the bath. The probability of channel opening was collected in the control and during cell exposure to different concentrations (0.3-100 μM) of VGB; and the values collected were then compared with those attained after the addition of TRAM-34 (3 μM). TRAM-34 is a known selective blocker of IKCa channels. The VGB concentration required to suppress 50% of channel activity was determined with the goodness of fits by means of a Hill function:
where IC50 or nH is the concentration required for a 50% inhibition or the Hill coefficient, respectively; [C] denotes the VGB concentration; and Emax indicates the maximal reduction of channel open probability (i.e., TRAM-34-sensitive channel activity) caused by VGB [20, 21].
Statistical analyses
For linear or nonlinear curve-fitting (e.g., exponential or sigmoidal curve), we appropriately fitted the data sets collected by using either Microsoft Excel® (Redmond, WA) or OriginPro 2016 (Microcal). Values are presented as the mean ± standard error of the mean (SEM) with sample sizes (n) indicating the number of 13-06-MG cells from which the experimental data was collected. The Student’s t-test or one-way analysis of variance (ANOVA) followed by a post-hoc Fisher’s least-significant difference test, were performed to analyze multiple groups. The data were further examined using a non-parametric Kruskal-Wallis test, subject to possible violation in the normality underlying ANOVA. Differences were considered statistically significant when the P-value was below 0.05.